Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (13): 2654-2666.doi: 10.3864/j.issn.0578-1752.2022.13.014

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Effects of lncFAM200B on the Lipid Deposition in Intramuscular Preadipocytes of Yak

RAN HongBiao(),ZHAO LiLing,WANG Hui(),CHAI ZhiXin,WANG JiKun,WANG JiaBo,WU ZhiJuan,ZHONG JinCheng()   

  1. Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Sichuan Province and Ministry of Education, Southwest Minzu University, Chengdu 610041
  • Received:2021-05-12 Accepted:2021-12-16 Online:2022-07-01 Published:2022-07-08
  • Contact: Hui WANG,JinCheng ZHONG E-mail:haluxi_7@163.com;wanghui892321@sina.cn;zhongjincheng518@126.com

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Abstract:

【Objective】The aim of this study was to analyze the effects of lncFAM200B in the lipid deposition in intramuscular preadipocytes of yak, which laid a foundation for further mechanism research. 【Method】 The longissimus dorsi muscle tissue of yak was collected and used to separate intramuscular preadipocytes. The adenovirus mediated overexpression technology was used to realize the overexpression of lncFAM200B, and the siRNA interference technology was used to analyze the function of lncFAM200B. The mRNA expression level of fat differentiation marker (PPARγ, C/EBPα and AP2) and the potential target (SIRT1 and PTEN) genes of lncFAM200B were detected via real-time fluorescent quantitative PCR (RT-qPCR). Oil red O staining, triacylglycerol (TAG) determination and CCK-8 determination methods were used to detect intracellular lipid droplet deposition and preadipocyte proliferation.【Result】The overexpression of lncFAM200B not only significantly increased the fat differentiation genes (C/EBPα and AP2, P<0.05) expression level, but also increased the lipid droplets deposition with large lipid droplets in the cells during induced differentiation. Conversely, lncFAM200B interference reduced the expression of PPARγ, C/EBPα and AP2 (P<0.05), and lipid droplet deposition. Furthermore, the triacylglycerol content after lncFAM200B overexpression 4 days was significantly higher than that of the control group (P<0.05), but lower after siRNA interference 6 days. Moreover, the SIRT1 levels increased with time was first decreased and then increased trend, and the PTEN had the opposite trend during lncFAM200B overexpression, and the opposite results of the two genes obtained during lncFAM200B interference. In addition, the results of CCK-8 experiment showed that there was a significant difference in cell proliferation activity both in overexpression or interference group after 72 hours. 【Conclusion】lncFAM200B could regulate yak adipose differentiation by influencing the expression of fat differentiation marker (C/EBPα and AP2), and further affect the triacylglycerol content and the lipid droplet deposition, but the detail mechanism need to be further research efforts.

Key words: yak, lncFAM200B, intramuscular preadipocytes, overexpression, interference

Table 1

The primer sequence for RT-qPCR"

基因名称 Gene name 登录号 Login ID 引物序列 (5′-3′) Primer sequence (5′-3′) 产物长度 Product size (bp)
lncFAM200B F:GCTTCCCATCAGAAAGTATCAGG
R:TTGTGTTGGTAGCTTGACTACG		 141
GAPDH NM_001034034.2 F:CCACGAGAAGTATAACAACACC
R:GTCATAAGTCCCTCCACGAT		 120
SIRT1 XM_024986766.1 F:TGGGGTTTCTGTTTCTTGTGG
R:CTTGAGGATCTGGAAGGTCTGG		 98
PTEN NM_001319898.1 F:GGAAAGGGACGAACTGGTGTAA
R:TGTCTCTGGTCCTTACTTCCCC		 109
AP2 XM_005897260 F:GGAAAGTCAAGAGCATCGTAAAC
R:CACCATCTTATCATCCACGAGTT		 110
C/EBPα NM_176784.2 F:GTGGACAAGAACAGCAACGAGTA
R:GCGGTCATTGTCACTGGTCAG		 138
PPARγ NM_181024.2 F:CGTGGACCTTTCTATGATGGA
R:GCTCTTGGGAACGGAATG		 117

Fig. 1

Cell adhesion (100×) A: Cell adherent after 1 h; B: Cell adherent after 24 h; C: Cell adherent after 6 days; D: Cell adherent after 8 days"

Fig. 2

The results of Oil Red O staining at different time after preadipocyte differentiation (200×) A: Differentiation day 0, B: Differentiation day 2, C: Differentiation day 4, D: Differentiation day 6, E: Differentiation day 8; F: Absorbance of Oil Red in different differentiation time. The same lowercase letter in picture F indicates that the difference is not significant (P>0.05), the different lowercase letters indicate significant differences (P<0.05). The same as Fig.3"

Fig. 3

Detection of triglyceride in preadipocyte differentiation at different time"

Fig. 4

RT-qPCR results of preadipocyte differentiation at different time points"

Fig. 5

Screening of experimental conditions A: Verification of restriction endonuclease digestion of overexpression vector, M: 1kb plus DNA Ladder; 1: Single enzyme digestion of PacⅠ; 2: Intact plasmid. B: Fluorescence detection of preadipocytes transfected with different MOI values(100×). C: Interference efficiency determination of siRNAs, the same letter indicates that the difference is not significant (P>0.05), the different letters indicate significant differences (P<0.05)"

Fig. 6

Effect of lncFAM200B overexpression on the expression of genes involved in lipid metablosim"

Fig. 7

Effect of lncFAM200B overexpression on the lipid droplet deposition A: The effects of lncFAM200B overexpression on lipid deposition(200×), a: Negative control group; b: Overexpression group. B: Detection of Oil Red absorption at different time after overexpression"

Fig. 8

The changes of triglyceride content after lncFAM200B overexpression"

Fig. 9

Effect of lncFAM200B interference on the expression of target genes"

Fig. 10

Effect of lncFAM200B interfering on the lipid droplet deposition A: The effects of lncFAM200B interfering on lipid deposition(200×), a: negative control group; b: interfering group. B: Detection of Oil Red absorption at different time after interfering"

Fig. 11

Detection of triglyceride content in cells after lncFAM200B interference"

Fig. 12

CCK-8 detection results A: Overexpression results; B: siRNA interference results. None * indicates the difference is not significant (P>0.05), * indicate significant differences (P<0.05)"

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